Anti-freeze proteins (AFPs) have been suggested for improving the freezing tolerance of foodstuffs. For the purpose of the invention, the term AFP has the meaning as well-known in the art, namely those proteins which exhibit the activity of inhibit the growth of ice crystals. See for example U.S. Pat. No. 5,118,792.
WO 90/13571 discloses antifreeze peptides produced chemically or-by recombinant DNA techniques. The AFPs can suitably be used in food-products. Example 3B shows modified ice crystal shapes if a water-ice mixture is frozen into a film in combination with 0.01 wt % of AFP.
WO 92/22581 discloses AFPs from plants which can be used for controlling ice crystal shape in ice-cream. This document also describes a process for extracting a polypeptide composition from extracellular spaces of plants by infiltrating leaves with an extraction medium without rupturing the plants.
WO 94/03617 discloses the production of AFPs from yeast and their possible use in ice-cream. WO 96/11586 describes fish AFPs produced by microbes.
Several literature places also mention the isolation and/or use of plant proteins for cryoprotection. Cryoprotective proteins have a function in the protection of plant membranes against frost damage. These proteins, however, do not possess recrystallisation inhibition properties and are, therefore, not embraced within the terms AFPs.
Hincha in Journal of Plant Physiology, 1992, 140, 236-240 describes the isolation of cryoprotective proteins from cabbage. Volger in Biochimica et Biophysica Acta, 412 (1975), 335-349 describes the isolation of cryoprotective leaf proteins from spinach. Boothe in Plant Physiol (1995), 108: 759-803 describes the isolation of proteins from Brassica napus. Again, these proteins are believed to be cryoprotective proteins rather than AFPs. Neven in Plant Molecular Biology 21: 291-305, 1993 describes the DNA characterisation of a spinach cryoprotective protein. Salzman in Abstracts and Reviews of the 18th Annual Meeting of the ASEV/Eastern Section in Am. J. Enol. Vitic., Vol. 44, No. 4, 1993 describes the presence of boiling-stable polypeptides in buds of Vitis. Although the proteins are analogous to fish antifreeze peptides, they are cryoprotective proteins and not AFPs. Lin in Biochemical and Biophysical Research Communication, Vol. 183, No. 3, 1992, pages 1103-1108 and in Lin, Plant Physiology (1992) 99, 519-525 describes the 15 kDa cryoprotective polypeptide from Arabidopsis Hakaira. Houde in The Plant Journal (1995) 8(4), 583-593 mentions cryoprotective proteins from wheat.
Up till now, however the use of AFPs has not been applied to commercially available food products. One reason for this are the high costs and complicated process for obtaining AFPs. Another reason is that the. AFPs which until now have been suggested for use in frozen food products cannot be incorporated in the standard formulation mix, because they tend to destabilise during processing especially during the pasteurisation step. This destabilisation is believed to be caused by the denaturation of the AFPs; this is a well-known effect commonly observed for peptides and proteins.
In our non pre-published patent application: PCT/EP97/03634 it has been described that AFPs can be isolated from natural sources such as cold-acclimatised grass by means of a new relatively simple process. This process leads for the first time to the identification of AFPs which can conveniently be incorporated in a mix for the preparation of frozen products before the pasteurisation thereof.
This process for the recovery of AFPs from grass involves the steps of                a) isolating a AFP containing juice from the grass;        b) heat treating the grass or the AFP containing juice to a temperature of at least 60° C.;        c) removing the insoluble fraction.        
Step c of the above process will usually take place after steps a and b. Step a and b can be done in any desired order, for example step a followed by step b (in that case the AFP rich juice will be heated) or step b followed by step a (in that case the natural source will be heated) or step a and b simultaneously.
This process has a number of advantages. Firstly by using the process it is no longer necessary to avoid rupturing of the grass such as required in the processes according to WO 92/22581. This immediately significantly increases the commercial applicability of the process, for example as compared to WO 92/22581, because high investment costs for specific processing are no longer necessary. Also by using the high temperatures it seems possible to extract from a large group of peptides present in the grass a very active AFP which is very active with respect to ice-recrystabisaffon inhibiton properties. Thirdly, contrary to expectations, the use of high temperatures does not denature all the proteinaceous material, but does only seem to denature some of the proteins, while the remaining grass AFP have an increased temperature stability. This renders it possible to include the isolated AFPs in compositions which need to be subjected to higher temperatures e.g. a pasteurisation step. This is especially surprising, because for example the AFPs from WO 92/22581 appear not stable under heating conditions.
The process as described above includes in step b the heating of the grass or the AFP rich juice to a temperature of more than 60° C. Preferably the temperature is from 60 to 110° C., most preferably from 80 to 105° C. The heating step can take place after the isolation of the protein rich juice (step a) or before the isolation of the protein rich juice. Any suitable way to heat the juice can be used, for example conventional or microwave heating, heating optionally with an added extraction medium, steaming etc.
If an extraction medium is used, preferably it is used in small volumes to avoid unnecessary dilution of the AFP fraction. Any suitable extraction medium can be used, although the use of water is especially preferred. If desired, additives may be added to the water prior to using it as an extraction medium. Most preferred, however water substantially free of additives is used.
Applicants have also found that by the above process a very active AFP derived from grass can be derived. For the purpose of the invention the term grass encompasses members of the Gramineae family including for example perennial grass such as Lolium perenne, Parapholis strigosa, Nardus stricta, Catapodium loliaceum and Lolium multiflorum, Poa trivialis, Poa pratensis and cereal crops such as winter rye, winter wheat and winter barley. Applicants have determined the amino acid sequence of this AFP.
Surprisingly it has been found that very active AFPs which can be derived from plants are characterised by a high level of the amino acids: Serine (S), Threonine (T) and Asparagine (N). In particular applicants have found that preferred AFPs of the invention are characterised in that at least 40% of the amino acids in the protein are selected from S, T and N. Preferably the AFPs are derived from grasses, especially the Gramineae family.
The preferred molecular weight of AFPs of the invention is from 8 to 16 kDa, more preferred 10-14 kDa, where this molecular weight is determined from the gene sequence or by mass spectrometry of the unmodified form e.g. in unglycosylated form.
A second aspect of the invention relates to an AFP which can be derived from grass, said AFP having an amino acid sequence (Seq. ID No. 1) from the N-terminus of:
D-E-Q-P-N-T-I-S-G-S-N-N-T-V-R-S-G-S-K-N-V-L-A-G-N-D-N-T-V-I-S-G-D-N-N-S-V-S-G-S-N-N-T-V-V-S-G-N-D-N-T-V-T-G-S-N-H-V-V-S-G-T-N-H-I-V-T-D-N-N-N-N-V-S-G-N-D-N-N-V-S-G-S-F-H-T-V-S-G-G-H-N-T-V-S-G-S-N-N-T-V-S-G-S-N-H-V-V-S-G-S-N-K-V-V-T-D-A
Also embraced in the scope of our invention are proteins having a sequence which has a high degree of similarity with the above sequence. For the purpose of the invention all RI active proteins having an amino acid sequence of at least 80% overlap with the above sequence are also embraced in the scope of the invention. More preferred is an overlap of at least 90%, most preferred more than 95%, e.g. those amino acid sequences which differ none or only one or two amino acids with the above sequence. Also isoforms of the above protein are embraced within the invention.
Also embraced within the scope of the present invention are modified versions of the above described proteins whereby said modification does not materially affect the ice recrystallisation inhibition properties, such as glycosylated versions thereof.
The AFP rich juice can be separated from the grass by any convenient process for example pressing, filtering, homogenising, extraction etc. Preferably the grass is made into small pieces or into a slurry before the protein rich fraction is collected, for example by filtering. This maceration can be done by any suitable method, for example in a blender. It will be well within the ability of the skilled person to divide the material into such a form that collection of the protein rich juice can readily take place.
After collecting and heating (in the desired order) the protein fraction the resulting AFP containing sample can then be treated by any convenient process in order to remove the insoluble fraction and retain the AFP rich liquid fraction. The insoluble fraction can be removed e.g. by filtering, precipitation etc. The AFP rich liquid can then advantageously be further processed to concentrate or isolate the AFPs to bring them in a form suitable for further use. Examples of suitable processes are drying to obtain a powder or paste, further concentration to obtain an AFP concentrate, chromatography to separate the AFPs from the extraction medium etc. Again it will be well within the ability of the skilled person to determine the suitable means and conditions for appropriate isolation.
Applicants have also determined the nucleic acid sequence that encodes the above described AFP. Accordingly a second aspect of the invention relates to a nucleic add sequence capable of encoding for the AFPs of the invention. Preferably said nucleic add has the sequence (Seq. ID No. 2) of:
GAT GAA CAG CCG AAT ACG ATT TCT GGG AGC AAC AAT ACT GTC AGATCC GGG AGC AAA AAT GTT CTT GCT GGG AAT GAC AAC ACC GTC ATATCT GGG GAC AAC AAT AGT GTG TCT GGG AGC AAC AAC ACT GTC GTAAGT GGG AAT GAC AAT ACC GTA ACC GGC AGC AAC CAT GTC GTA TCAGGG ACA AAC CAT ATC GTT ACA GAC AAC AAC AAT AAC GTA TCC GGGAAC GAT AAT AAT GTA TCC GGG AGC TTT CAT ACC GTA TCC GGG GGGCAC AAT ACT GTG TCC GGG AGC AAC AAT ACC GTA TCT GGG AGC AACCAC GTT GTA TCT GGA AGC AAC AAA GTC GTG ACA GAC GCT TAA
Also embraced within the scope of the present invention are alleles or other nucleic acid sequences which are capable to encode the above described AFPs, for example those nucleic acid sequences wherein wrt the above sequence one or more codons have been replaced by their synonyms (i.e. codons encoding for the same amino acid).
Vectors containing a nucleic acid sequence capable of encoding the AFP of the invention are also embraced within the scope of the invention.
Based on the above information it is also possible to genetically modify other natural sources such that they produce the advantageous AFP as identified here-above.
Applicants also have found that AFPs of the above sequence have improved ice-recrystallisation inhibition properties. A suitable test for determining the ice recrystallisation inhibition properties is described in the examples and involves the quick freezing to −40° C. follow by storage for one hour at 60° C. Preferably AFPs which are subject to this test after heat-treatment result in an ice crystal particle size which is less than 5 μm larger than the ice crystal size of a sample with the same AFP which was not heat-treated. Preferably the difference is less than 3 μm, most preferred less than 1 μm.
Preferably those AFPs are chosen which have significant ice-recrystallisation inhibition properties. A suitable test for determining the recrystallisation inhibition properties is indicated in the examples. Preferably AFPs in accordance to the invention provide a ice particle size following an ice recrystallisation inhibition assay as described in the examples of 15 μm or less, more preferred from 5 to 15 μm.
The AFP of the invention can conveniently be used in food products, preferably in food products which are frozen or intended to be frozen. Especially preferred is the use of AFPs in products which are heated e.g. by pasteurisation, blanching or sterilisation prior to freezing. Especially preferred is the use in frozen confectionery products.
Examples of such food products are: frozen confectionery mixes such as ice-cream mixes and water-ice mixes which are intended to be pasteurised prior to freezing. Such mixes are usually stored at ambient temperature. Suitable product forms are for example: a powder mix which is packed for example in a bag or in sachets. Said mix being capable of forming the basis of the frozen food product e.g. after addition of water and optionally other ingredients and -optional-aeration.
Another example of a suitable mix could be a liquid mix (optionally aerated) which, if necessary after addition of further components and optional further aeration can be frozen.
The clear advantage of the above mentioned mixes is that the presence of the AFP ingredient makes that the mixes can be frozen under quiescent conditions, for example in a shop or home freezer without the formation of unacceptable ice crystal shapes and hence with a texture different to products normally obtained via quiescent freezing.
Very conveniently these mixes are packed in closed containers (e.g. cartons, bags, boxes, plastic containers etc). For single portions the pack size will generally be from 10 to 1000 g. For multiple portions pack sizes of up to 500 kg may be suitable. Generally the pack size will be from 10 g to 5000 g.
As indicated above the preferred products wherein the AFPs are used are frozen confectionery product such as ice-cream or water-ice. Preferably the level of AFPs is from 0.00001 to 0.5 wt % based on the final product. If dry-mixes or concentrates are used, the concentration may be higher in order to ensure that the level in the final frozen product is within the above ranges.
For the purpose of the invention the term frozen confectionery product includes milk containing frozen confections such as ice-cream, frozen yoghurt, sherbet, sorbet, ice milk and frozen custard, water-ices, granitas and frozen fruit purees. For some applications the use in fermented food products is less preferred.
Preferably a the level of solids in the frozen confection (e.g. sugar, fat, flavouring etc) is more than 4 wt %, for example more than 30 wt %, more preferred from 40 to 70wt %.
Frozen confectionery products according to the invention can be produced by any method suitable for the production of frozen confectionery. Especially preferably however all the ingredients of the formulation are fully mixed before pasteurisation and before the freezing process starts. The freezing process may advantageously involve a hardening step, for example to a temperature of −30 Celsius or lower.